IFN-β2/IL-6 receptor its preparation and pharmaceutical compositions containing it

ABSTRACT

Human natural IFN-β2/IL-6 receptor and its soluble extracellular fragment are provided in substantially purified form and are shown to stimulate and to enhance beneficial effects of IFN-β2/IL-6, such as its antiproliferative activity. Polyclonal and monoclonal antibodies raised against the soluble fragment of the receptor are also provided.

FIELD OF THE INVENTION

This invention relates to substantially purified humanInterferon-β2/Interleukin-6 Receptor (hereinafter IFN-β2/IL-6-R), itssoluble extracellular fragment, salts, functional derivatives,precursors and active fractions thereof. The invention also relates to aprocess for the purification of human natural IFN-β2/IL-6 R and itssoluble extracellular fragment, to the cloning of the solubleextracellular fragment and its production by recombinant DNA techniques,and to polyclonal and monoclonal antibodies raised thereto. It furtherrelates to pharmaceutical compositions comprising human naturalIFN-β2/IL-6-R or its soluble extracellular fragment, or salts,functional derivatives, precursors and active fractions thereof.

BACKGROUND OF THE INVENTION

Interferon-β2, now designated Interleukin-6 (hereinafter IFN-β2/IL-6),is a multifunctional cytokine that regulates the growth anddifferentiation of various cells and tissues and appears to be one ofthe important mediators of the response to viral and bacterialinfections and to shock. The biological effects now associated withIFN-β2/IL-6 include: stimulation of immunoglobulin secretion by mature Blymphocytes (BSF-2 activity), growth stimulation of plasmacytomas andhybridomas (HGF activity), activation of T cells, stimulation of hepaticacute phase protein synthesis (HSF activity), stimulation ofhematopoiesis, cell differentiation (DIF activity), inhibition of tumorcell growth (AP activity) and other IFN-like effects. As a typicalcytokine, IFN-β2/IL-6 is secreted by many cell types and acts in variouscombinations with other interleukins and interferons. Among itsactivities viewed as having antitumor potential are: inhibition ofcancer cell growth and colony formation, differentiation of malignantcells to more normal phenotypes, stimulation of normal hematopoiesis,stimulation of T-lymphocyte activation, stimulation of antibodysecretion by B-cells, stimulation of complement synthesis, andstimulation of antiprotease synthesis.

The cloning of the human IFN-β2/IL-6 receptor was reported (Yamasaki, etal., Science, Vol. 241, pp. 825-828). However, the natural humanIFN-β2/IL-6 receptor and a soluble extracellular fragment thereof havenot been isolated and described in the literature.

SUMMARY OF THE INVENTION

It has now been discovered that the IFN-β2/IL-6-R and a soluble fragmentthereof strongly enhance the stimulatory effect of IFN-β2/IL-6 on mouseplasmacytoma cells and markedly enhance the growth inhibitory effect ofhuman IFN-β2/IL-6 on human breast cancer cells and thus can be used toenhance the biological activity of IFN-β2/IL-6. Thus, the presentinvention provides human natural Interferon-β2/Interleukin-6 Receptor,herein designated IFN-β2/IL-6-R, its soluble extracellular fragment,salts, functional derivatives, precursors and active fractions thereof,and mixtures of any of the foregoing, which specifically bindIFN-β2/IL-6 and can stimulate and enhance the beneficial biologicaleffects of IFN-β2/IL-6.

The invention is directed to said human natural IFN-β2/IL-6-R insubstantially purified form, being free of proteinaceous impurities, andto a process for its isolation.

The invention also relates to the human IFN-β2/IL-6-R solubleextracellular fragment in substantially purified form, being free ofproteinaceous impurities and moving as a single peak on reversed-HPLCand to a process for the isolation and purification of said fragmentfrom human fluids, e.g. urine.

The invention is also directed to polyclonal and monoclonal antibodiesagainst the human IFB-β2/IL-6-R and its soluble extracellular fragment.

The invention further concerns the production of the IFN-β2/IL-6-Rsoluble extracellular fragment by recombinant DNA techniques, includingthe preparation of DNA sequences coding for the IFN-β2/IL-6-R solubleextracellular fragment, or for a protein substantially homologoustherewith, the construction of expression vehicles comprising them andof host cells transformed therewith, and to a process for producingIFN-β2/IL-6-R soluble extracellular fragment or a protein substantiallyhomologous therewith, by culturing said transformant cells in a suitableculture medium.

The human natural IFN-β2/IL-6-R of the invention and its solubleextracellular fragment and salts, functional derivatives, precursors andactive fractions thereof, and mixtures of any of the foregoing, are foruse as active ingredients in combination with IFN-β2/IL-6 ofpharmaceutical compositions to enhance the beneficial effects ofIFN-β2/IL-6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the reversed-phase HPLC column elution pattern ofIFN-β2/IL-6-R soluble extracellular fragment fractions after partialpurification on an immobilized IFN-β2/IL-6 column.

FIG. 2 shows the results of analysis of the purified IFN-β2/IL-6-Rsoluble extracellular fragment fraction by SDS-PAGE under non-reducingconditions, followed by silver staining (lanes 1 and 2), or followed byelectroblotting, ¹²⁵ I-rIFN-β2/IL-6 binding, and visualization byautoradiography (lanes 3 and 4).

FIG. 3 shows the enhancement of IFN-β2/IL-6 HGF activity byIFN-β2/IL-6-R soluble extracellular fragment on human breast carcinomaT47D cells.

DETAILED DESCRIPTION OF THE INVENTION

The receptors of IFN-β2/IL-6 on various human cells are identified bycross-linking experiments with radiolabelled IFN-β2/IL-6. Briefly pureIFN-β2/IL-6 is labelled with [¹²⁵ I] by the chloramine-T methodaccording to published procedures, retaining its intact biologicalactivity. Such labelled IFN-β2/IL-6 is allowed to react with varioushuman cells at 4° C. and the resulting IFN-β2/IL-6 receptor complexesare covalently cross-linked and then analyzed by polyacrylamide gelelectrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS)followed by autoradiography. After their identification, the receptorsare isolated by a method which comprises solubilizing human cellsbearing the receptors to obtain a suspension, centrifuging thesuspension to obtain a supernatant, applying the supernatant to animmobilized IFN-β2/IL-6 or anti-IFN-β2/IL-6-R monoclonal antibodycolumn, and eluting the bound receptor protein by varying pH conditions,in a state of enhanced purity. If necessary, the eluted fractions may befurther purified.

The soluble extracellular fragment of IFN-β2/IL-6-R of the invention wasfound in human urine. In its substantially purified form, which issubstantially free of proteinaceous impurities, it has a molecularweight of about 50 (40-60) K when analyzed by SDS PAGE undernon-reducing conditions and it moves as a single peak on reversed-phaseHPLC.

It is further characterized by the following sequence obtained byN-terminal sequence analysis of the protein: ##STR1##

The present invention encompasses a polypeptide comprising the abovesequence as well as any other polypeptide in which one or more aminoacids in the structure of the IFN-β2/IL-6-R fragment are deleted orreplaced with other amino acids, or one or more amino acids are addedthereto, as long as they bind specifically IFN-β2/IL-6-R.

This invention also relates to a process for isolating IFN-β2/IL-6-Rsoluble extracellular fragment from a human fluid, e.g. urine, and itspurification. In one preferred embodiment, the substantially purifiedreceptor fragment of the invention is produced by a process whichcomprises:

a) recovering the crude protein fraction from a dialyzed concentrate ofhuman urine of healthy donors;

b) subjecting said crude protein fraction of step (a) to affinitypurification on a column of immobilized IFN-β2/IL-6;

c) applying said affinity purified active fractions of IFN-β2/IL-6binding proteins from step (b) to reversed-phase HPLC; and

d) recovering the fractions eluting at 39% acetonitrile and containingsubstantially purified IFN-β2/IL-6-R soluble extracellular fragment,said protein having a molecular weight of about 50K on SDS PAGE undernon-reducing conditions and moving as a single peak on reversed-phaseHPLC.

In a preferred embodiment, the crude protein fraction of step (a) issubjected first to ion exchange chromatography, for example, on acarboxymethyl Sepharose (CM-Sepharose or CMS) column.

In another preferred embodiment, the affinity purification of step (b)is performed on a column of immobilized monoclonal antibodies raisedagainst the IFN-β2/IL-6-R soluble fragment. The invention furtherrelates to the preparation of IFN-β2/IL-6-R soluble extracellularfragment by genetic engineering techniques and encompasses all the toolsused in these techniques. Thus the invention concerns DNA moleculescomprising the nucleotide sequence coding for said receptor fragment orfor a protein substantially homologous therewith. These DNA moleculesmay be genomic DNA, cDNA, synthetic DNA and combinations thereof.

The cloning of the IFN-β2/IL-6-R soluble extracellular fragment may becarried out by different techniques. The DNA coding for the fragment issynthesized or the gene coding therefor is isolated from a DNA libraryor the gene coding for the whole IFN-β2/IL-6 receptor is obtained firstand then cut by known techniques. According to one approach, specificantibodies (polyclonal or monoclonal) to IFN-β2/IL-6 receptor areproduced and used to search for cells producing the receptor byimmunofluorescence or by Western blot. Then, mRNA is extracted fromthese IFN-β2/IL-6 receptor producing cells and is converted to cDNA bycontacting with reverse transcriptase for a time and under conditionssuitable to form said cDNA. The cDNA is cloned in an expression vectorsuch as lambda gt 11, and screened by the use of the antibodies. Thelambda gt 11 expression vector can be used for insertion of DNA up to 7kb in length at a unique EcoRI site 53 bases upstream from theβ-galactosidase termination codon. Therefore, foreign sequences DNA maybe inserted into this site and expressed under appropriate conditions asfusion proteins. The lambda gt 11 expression vector is particularlyuseful for the construction of cDNA libraries to be screened withantibody probes (Huynh, T.V. et al. in: David Glover (ed.), DNA CloningTechniques: A Practical Approach, IRL Press, Oxford (1984) pp. 49-78).

Following another approach, a synthetic oligonucleotide or a mixture ofsynthetic oligonucleotides, whose sequence is derived from the aminoacid sequence of a fragment of the protein, e.g., the N-terminal aminoacid sequence, are produced and used as probes for cloning the cDNA orthe genomic DNA coding for the IFN-β2/IL-6 receptor or for its solublefragment. Suitable DNA preparations, such as human genomic DNA, areenzymatically cleaved by restriction enzymes, or randomly sheared, andthe fragments inserted into appropriate recombinant vectors to form agene library. Such vectors can then be screened with syntheticoligonucleotide probes in order to identify a sequence coding for theIFN-β2/IL-6 receptor or for its soluble fragment.

Alternatively, the mRNA is isolated from cells which express thereceptor and is converted, after purification, to cDNA as describedabove. The cDNA is converted to double-stranded cDNA by knowntechniques, is cloned and the resulting clones are screened with anappropriate probe for cDNA coding for the desired sequences. Once thedesired clone is isolated, the cDNA is manipulated in substantially thesame manner as the genomic DNA. However, with cDNA there will be nointrons or intervening sequences.

The invention also relates to synthetic oligonucleotides to be used asprobes to the DNA coding for IFN-β2/IL-6-R or for its soluble fragment.They are synthesized by known methods on the basis of the amino acidsequence of fragments of IFN-β2/IL-6-R. For this purpose, it is possibleeither to perform sequence analysis of the intact IFN-β2/IL-6-R or ofits soluble extracellular fragment to obtain peptide fragments thereofand to characterize their amino acid sequence. The peptide fragments areobtained by subjecting purified protein preparations to fragmentation,e.g. by digestion with proteases such as trypsin, chymotrypsin or papainby methods well known in the art (Oike, Y. et al. (1982) J. Biol. Chem.257:9751-9758), they are then separated by reverse-phase HPLC andsequenced by automatic amino acid sequencing techniques.

Once one or more suitable peptide fragments have been sequenced or apartial sequence of the protein is determined, the DNA sequences capableof encoding them are examined. Due to the degeneration of the geneticcode, more than one codon may be used to encode a particular amino acidand one or more different oligonucleotides can be produced, each ofwhich would be capable of encoding the IFN-β2/IL-6-R peptide fragments(Watson, J. D., in: Molecular Biology of the Gene, 3rd ed., W. A.Benjamin, Inc. Menlo Park, CA (1977), pp. 356-357). However, only onemember of the set contains the nucleotide sequence that is identical tothe nucleotide sequence of the gene. Its presence within the set and itscapability to hybridize to DNA even in the presence of the other membersof the set, makes it possible to employ the unfractionated set ofoligonucleotides in the same manner in which one would employ a singleoligonucleotide to clone the gene that encodes the peptide. The use ofsuch oligonucleotide or set of oligonucleotides containing thetheoretical "most probable" sequence capable of encoding theIFN-β2/IL-6-R gene fragments (following the "codon usage rules"disclosed by Lathe, R., et al. (1985) J. Molec. Biol. 183:1-12) permitsto identify the sequence of a complementary oligonucleotide or set ofoligonucleotides which is capable of hybridizing to the "most probable"sequence encoding the IFN-β2/IL-6-R or at least a portion thereof, or aset of such sequences. This oligonucleotide containing such acomplementary sequence is then synthesized and employed as a probe toidentify and isolate a DNA molecule coding for the IFN-β2/IL-6-R of theinvention from a DNA library (Maniatis, T. et al. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY(1982).

In one of the embodiments, the isolation of the gene of the IFN-β2/IL-6receptor is done by colony hybridization techniques under stringentconditions. Procedures for hybridization of nucleic acids are commonknowledge and are disclosed, for example, in Maniatis, T., MolecularCloning: A Laboratory Manual, op. cit. and in Haymes, B. T., et al.,Nucleic Acid Hybridization: A Practical Approach, IRL Press, Oxford,England (1985). By hybridization with the above nucleotide or set ofoligonucleotides probes, it is possible to identify in a cDNA or genomiclibrary, the DNA sequences capable of such hybridization and they arethen analyzed to determine to what extent they contain encodingsequences for the IFN-β2/IL-6-R. The DNA coding for the receptor solubleextracellular fragment of the invention is then obtained from the DNA ofpositive clones of the whole receptor by known procedures and isinserted into appropriately constructed expression vectors by techniqueswell known in the art (see Maniatis et al., op cit.). Double-strandedcDNA is linked to plasmid vectors by homopolymeric tailing or byrestriction linking involving the use of synthetic DNA linkers orblunt-ended ligation techniques. DNA ligases are used to ligate the DNAmolecules and undesirable joining is avoided by treatment with alkalinephosphatase.

For expression of the desired protein, the expression vector shouldcomprise also specific nucleotide sequences containing transcriptionaland translational regulatory information linked to the DNA coding forthe desired protein in such a way as to permit gene expression andproduction of the protein. First, in order for the gene to betranscribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters) andare different for prokaryotic and eukaryotic cells. High levels of geneexpression in prokaryotic cells are achieved by using alsoribosome-binding sites, such as the Shine-Dalgarno sequence (SDsequence). For eukaryotic hosts, different transcriptional andtranslational regulatory sequences may be employed, depending on thenature of the host. They may be derived from viral sources, such asadenovirus, bovine papilloma virus, Simian virus, or the like, where theregulatory signals are associated with a particular gene which has ahigh level of expression. Examples are the TK promoter of Herpes virus,the SV40 early promoter, the yeast ga14 gene promoter, etc.Transcriptional initiation regulatory signals may be selected whichallow for repression and activation, so that expression of the genes canbe modulated.

The DNA molecule comprising the nucleotide sequence coding for a proteincomprising the amino acid sequence of the IFN-β2/IL-6-R solubleextracellular fragment of the invention, preceded by a nucleotidesequence of a signal peptide and the operably linked transcriptional andtranslational regulatory signals, is inserted into a vector which iscapable of integrating the desired gene sequences into the host cellchromosome. The cells which have stably integrated the introduced DNAinto their chromosomes can be selected by also introducing one or moremarkers which allow for selection of host cells which contain theexpression vector.

In a preferred embodiment, the introduced DNA molecule will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Prokaryotic and eukaryotic plasmidsare well-known from the literature. Factors of importance in selecting aparticular plasmid or viral vector include the ease with which recipientcells that contain the vector may be recognized and selected from thoserecipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it isdesirable to be able to "shuttle" the vector between host cells ofdifferent species.

Once the vector or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc. Host cells to be used in this invention may be either prokaryoticor eukaryotic. Preferred prokaryotic hosts include bacteria such as E.coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Under such conditions,the protein will not be glycosylated. The prokaryotic host must becompatible with the replicon and control sequences in the expressionplasmid. Preferred eukaryotic hosts are mammalian cells, e.g., human,monkey, mouse and chinese hamster ovary (CHO) cells, because theyprovide post-translational modifications to protein molecules, includingcorrect folding or glycosylation at correct sites. Also yeast cells cancarry out post-translational peptide modifications, includingglycosylation.

After the introduction of the vector, the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired IFN-β2/IL-6-R fragment. The expressed proteinis then isolated and purified in accordance with the purification methoddescribed in the present application or by any other conventionalprocedure involving extraction, precipitation, chromatography,electrophoresis, or the like.

A further purification procedure that may be used in preference forpurifying the protein of the invention is affinity chromatography usinganti-IFN-β2/IL-6-R monoclonal antibodies, which are produced andimmobilized on a gel matrix contained within a column. Impurepreparations containing the recombinant protein are passed through thecolumn. The protein will be bound to the column by the specific antibodywhile the impurities will pass through. After washing, the protein iseluted from the gel by a change in pH or ionic strength.

As used herein the term `salts` refers to both salts of carboxyl groupsand to acid addition salts of amino groups of the protein moleculeformed by means known in the art. Salts of a carboxyl group includeinorganic salts, for example, sodium, calcium, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine. Acid addition salts include, forexample, salts with mineral acids and salts with organic acids.

"Functional derivatives" as used herein covers derivatives which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e. they do not destroy the activity of the protein and donot confer toxic properties on compositions containing it. Thesederivatives include aliphatic esters or amides of the carboxyl groups,and N-acyl derivatives of free amino groups or O-acyl derivatives offree hydroxyl groups formed with acyl moieties (e.g. alkanoyl orcarbocyclic aroyl groups).

"Precursors" are compounds formed prior to, and converted into,IFN-β2/IL-6-R in the animal or human body. As "active fractions" of thesubstantially purified protein, the present invention covers anyfragment or precursors of the polypeptide chain of the protein moleculealone or together with associated molecules or residues linked thereto,e.g. sugar or phosphate residues, or aggregates of the protein moleculeor the sugar residues by themselves, provided said fraction has theability to inhibit the cytotoxic effect of TNF on cells and/or tomaintain its prolonged beneficial effect.

The invention further relates to antibodies against IFN-β2/IL-6-R andits soluble extracellular fragment and to F(ab) fragments thereof. Thefunctional interaction of the antibodies of the present invention withIFN-β2/IL-6-R provides also a new diagnostic tool, based on immunoassayssuch as radioimmunoassay, ELISA etc., for the detection of over- orunder-production of IFN-β2/IL-6-R by cells in the body in certaindisorders.

The antibodies may be either polyclonal or monoclonal. They may beraised in rabbits, mice or other animals or tissue cultured cellsderived thereof or can be products of cells of human origin. They mayalso be produced by recombinant DNA technology either in a formidentical to that of the native antibody or as chimeric molecules,constructed by recombination of antibody molecules of man and animalorigins. The development of antibody level is followed by the ability ofthe animal serum to inhibit the hybridoma growth factor (HGF) activityof IFN-β2/IL-6.

For the preparation of the antibodies, either purified IFN-β2/IL-6receptor or its soluble extracellular fragment or one or more syntheticpeptides identical to the known sequence of the proteins, e.g. to theN-terminal protein sequence, may be used to immunize animals. A furtherpossibility is to fuse one of the possible nucleotide sequences codingfor a fragment of the receptor to the gene coding for Protein A, toexpress the fused Protein A-IFN-β2/IL-6-R gene in E. coli, to purify thefused protein by affinity chromatography on IgG Sepharose column andthen to use it to immunize animals.

The monoclonal antibodies of the present invention are prepared usingconventional hybridoma technique (Kohler et al. (1975) Nature 256:495;Kohler et al. (1976) Eur. J. Immunol. 6:511). After immunization, spleencells alone and/or together with lymph node cells of the immunizedanimals are isolated and fused with a suitable myeloma cell line. Afterfusion, the resulting hybridoma cells are selectively maintained in HATmedium and then cloned. The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding IFN-β2/IL-6 receptor or its soluble extracelularfragment. After identification, the desired clones are grown in bulk,either in suspension culture or in ascitic fluid, by injecting the cellsinto the peritoneum of suitable host mice. The monoclonal antibodiesproduced by the hybridomas are then isolated and purified.

As mentioned before, the monoclonal antibodies may also be immobilizedand used for the purification of the IFN-β2/IL-6 receptor or its solubleextracellular fragment in affinity purification procedure using animmunoadsorbent column.

The IFN-β2/IL-6-R and its soluble extracellular fragment and salts,functional derivatives, precursors and active fractions thereof, andmixtures of any of the foregoing, are indicated for stimulating andenhancing the desirable effects of IFN-β2/IL-6 in mammals, such as theantiproliferative activity of IFN-β2/IL-6.

The present invention further relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier, IFN-β2/IL-6 and theIFN-β2/IL-6-R, its soluble extracellular fragment or salts, functionalderivatives, precursors or active fractions thereof, and mixtures of anyof the foregoing, as active ingredients. These compositions may be usedin any condition where it is desired to stimulate the activity of IL-6.Of course the amount of IFN-β2/IL-6 to be administered is lower in viewof the enhancing effect of the receptor. The way of administration canbe via any of the accepted modes of administration for similar agentsand will depend on the condition to be treated.

The pharmaceutical compositions of the invention are prepared foradministration by mixing the active ingredients with physiologicallyacceptable carriers, stabilizers and excipients, and prepared in dosageform, e.g. by lyophilization in dosage vials. The amount of activecompounds to be administered will depend on the route of administration,the disease to be treated and the condition of the patient.

The IFN-β2/IL-6-R and its soluble extracellular fragment and salts,functional derivatives, precursors and active fractions thereof, andmixtures of any of the foregoing, may also be used alone for enhancingthe activity of endogenously formed IFN-β2/IL-6 in conditions likebacterial infections, burns, trauma, etc.

The invention will now be illustrated by the following non-limitingexamples:

EXAMPLE 1 Isolation and purification of IFN-β2/IL-6 receptor from humanplacenta

Placental membranes are prepared according to the method of R. A. Hockand M. D. Holleneberg (1980), J. Biol. Chem., 255, 10731-10736, asfollows: placenta pieces are homogenized in a buffer consisting of 25 mMTris-HCl, pH 7.4, 0.25M sucrose, 0.1M NaCl, 1.5 mM MgCl₂, 2 mM EGTA, 1mM PMSF and 22 Tiu/ml Aprotinin, followed by filtering through gauze andcentrifugation (10,000×g for 30 min. at 4° C.). NaCl and MgSO₄ are addedto the supernatant to a final concentration of 0.1M and 0.2 mMrespectively. The mixture is spun (48,000×g for 40 min. at 4° C.) andthe resulting pellet is resuspended in a buffer of 10 mM Hepes, pH 7.4,150 mM NaCl and 1 mM PMSF and 22 Tiu/ml. The membranes are thensolubilized in a solubilization buffer (final concentrations: 10 mMHepes, pH 7.4, 1-2% Triton X-100, 1 mM PMSF and 20 units/ml aprotinin).The suspension is spun first at 10,000×g for 15 min. and then at100,000×g for 60 min. The supernatant is applied to an immobilizedIFN-β2/IL-6 column (2,5 mg per 0.8 ml of Affigel-10). Loading is a flowrate of 0.2-0.5 ml/min. The column is then washed with PBS (50 ml) andthe bound material is eluted with a solution of 25 mM citric acid.Fractions of 1 ml are collected and immediately neutralized with 1MHepes, pH 8.5. Each fraction is tested for its ability to bind ¹²⁵I-IFN-β2/IL-6 and for protein content. Protein is determined withfluorescamine.

EXAMPLE 2 Isolation and purification of IFN-β2/IL-6-R solubleextracellular fragment

2.1 Preparation of the urine concentrate

A pool of 200 liter urine from healthy menopausal women was subjected tomicrofiltration on a Pellicon membrane with a pore size of 0.45 μm. Thefiltrate was concentrated by ultrafiltration using a Pellicon membranewith a molecular weight cut off of 10K to a final volume of 500 ml. Theconcentrate was dialyzed against phosphate buffered saline containing 1mM benzamidine and 0.1% sodium azide.

2.2 Ion-exchange chromatography on CM-Sepharose

A CM-Sepharose (Pharmacia, Uppsala, Sweden) cation exchange column(2.7×10 cm) was prewashed with 1M NaCl, 10 mM citric acid pH 5.0,containing 0.02% NaN₃ (buffer C) and equilibrated with 10 mM citric acid(pH 5) containing 0.02% NaN₃ (buffer A). The concentrate of urinaryproteins was dialyzed against buffer A, and centrifuged for 15 min. at8000×g. The supernatant was applied at 4° C. on the column at a flowrate of 2 ml/min. The column was washed with 1500 ml buffer A and elutedwith 250 ml of a solution containing 200 mM NaCl, 10 mM citric acid (pH5.0) and 0.02% NaN₃ (buffer B). A second step of elution was performedwith 150 ml buffer C. Fractions of 50 ml were collected and tested forIFN-β2/IL-6 binding activity (binding of ¹²⁵ I-IFN-β2/IL-6) and theirprotein concentration was determined.

2.3 Affinity purification on an IFN-β2/IL-6 column

IFN-β2/IL-6 was brought to a concentration of 5 mg/ml, then equilibratedwith PBS containing 0.02% sodium azide and coupled to Affigel-10 (2.5 mgto 0.8 ml beads). The concentrate of urinary proteins of step 2.1 or 2.2was equilibrated with phosphate-buffered saline (PBS) and applied to theIFN-β2/IL-6 Affigel-10 column (2.5 mg of protein bound to 0.8 ml ofAffigel-10) at a flow rate of 0.2 ml/min. at 4° C. Unbound proteins werewashed with PBS and the bound proteins were then eluted at pH 2.5 byapplying a solution of 25 mM citric acid, 1 ml fractions were collectedinto tubes containing 1M Hepes pH 8.5. The eluted protein was monitoredfor protein and for binding of ¹²⁵ I-IFN-β2/IL-6 followingelectroblotting.

2.4 Reversed-phase high pressure liquid chromatography

The reversed-phase HPLC column Aquapore RP-300 4.6×30 mm (Brownlee Labs)was prewashed with 0.3% aqueous trifluoroacetic acid (TFA) (Buffer F)until a stable baseline was obtained by the fluorescamine detectionsystem. The protein peak fractions eluted from the affinity IFN-β2/IL-6columns of step 2.3 were pooled and injected in one 1.8 ml portion ontothe column. The column was run with Buffer F at a flow rate of 0.5ml/minute until the fluorometer did not detect any protein. Elution wasperformed at a flow rate of 0.5 ml/minute, with a linear gradient ofacetonitrile in Buffer F (- - - ) (0-20% for 5 minutes, followed by20-50% for 60 minutes, and finally 50-80% for 5 minutes). The column wasthen washed for 15 minutes with 80% acetonitrile. Fractions of 0.5 mlwere collected and tested for protein content (--) and for binding of¹²⁵ I-IFN-β2/IL-6 following electroblotting. As shown in FIG. 1, theactive proteins were found to elute as one protein peak, in fractionscorresponding to about 39% acetonitrile.

2.5 SDS-PAGE and binding to ¹²⁵ I-IFN-β2/IL-6

In order to monitor the result of the purification, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performedunder non-reducing conditions according to the method of Laemmli U.K. etal. (Nature (1970) 227:680). Samples of the active fractions elutingfrom the reversed-phase HPLC, were mixed with 3×concentrated samplebuffer containing no SDS and no β-mercaptoethanol and loaded on a 12%acrylamide gel. In FIG. 2, as a reference for molecular weight, amixture of molecular weight markers (α lactalbumin 14.4K, soya beantrypsin inhibitor 20.1K, carbonic anhydrase 30K, ovalbumin 43K, bovineserum albumin 67K, and phosphorylase b. 94K) was loaded on lane 1. Thegel was run at 150 volt and the protein bands were visualized by silverstaining (Oakley, B. R. et al. Anal. Biochem. 105:361). In lane 2, it isshown that the HPLC-purified IFN-β2/IL-6 binding protein moved as asingle band, with an apparent molecular weight of 50 (40-60)K. Bindingof ¹²⁵ I-IFN-β 2/IL-6 (2.2×10⁷ cpm/μg, 1.5×10⁶ cpm/ml) was donefollowing SDS-PAGE under non-reducing conditions and electroblottingonto nitrocellulose membrane (Schleicher and Schuell 0.45 μm), performedessentially as the Western blotting method (Towbin, H. et al., Proc.Natl. Acad. Sci. USA, 76, 4350-4354, 1979). As shown in lane 3, only the50 (40-60)K protein of the partially purified protein sample from theIFN-β2/IL-6 affinity column eluate reacted specifically with ¹²⁵I-IFN-β2/IL-6. A purified IFN-gamma binding protein sample fromIFN-gamma affinity column eluate was used as a negative control (lane4). Lanes 3 and 4 were visualized by autoradiography.

2.6 N-Terminal Sequence Analysis

Samples of the substantially purified IFN-β2/IL-6-R solubleextracellular fragment of the invention (1-5 μg, 50-200 pmol each) wereapplied to pretreated, biobrene-coated glass-fiber discs. The drieddiscs were subjected to repetitive cycles of Edman degradation in anautomated pulsed liquid gas phase protein microsequencer (Model 475)with an on-line HPLC PTH-amino acid analyzer (Model 120) and a dataacquisition and processing unit Model 900, (all from Applied BiosystemsInc. Foster City, CA, U.S.A.). The computer-derived sequence wascompared with the raw data and was corrected when necessary. Altogethertwo separate analyses were performed in order to confirm the sequencedata. The initial yield was over 40%, indicating that the major proteinin the preparation (the 50K band) is related to the resulting sequence.The N-terminal sequencing of this soluble extracellular fragment of theIFN-β2/IL-6-R gave the following amino acid sequence: ##STR2##

EXAMPLE 3 Preparation of polyclonal antibodies to IFN-β2/IL-6-R

Rabbits were initially injected subcutaneously with 10 μg of a purepreparation of the IFN-β2/IL-6-R soluble extracellular fragmentemulsified in complete Freund's adjuvant. Three weeks later they wereinjected again subcutaneously with 10 μg of the preparation inincomplete Freund's adjuvant. Three additional injections as solution inPBS were given at 10 day intervals. The rabbits were bled 10 days afterthe last immunization. The development of antibody level was followed bythe ability of the rabbit serum to inhibit the HGF (hybridoma growthfactor) activity of IFN-β2/IL-6. The immunoglobulins of the rabbit serawere precipitated by the addition of ammonium sulfate andcentrifugation, and purified by dialysis and chromatography.

EXAMPLE 4 Preparation of monoclonal antibodies to IFN-β2/IL-6-R

Female Balb/C mice (3 months old) were first injected with 2.5 μgpurified IFN-β2/IL-6-R soluble extracellular fragment in an emulsion ofcomplete Freund's adjuvant, and three weeks later, subcutaneously inincomplete Freund's adjuvant. Three additional injections were given at10 day intervals, subcutaneously in PBS. A final boost was givenintraperitoneally 3 days before the fusion to the mouse showing thehighest binding titer in inverted solid phase RIA. Fusion was performedusing NSO/1 myeloma cell line and lymphocytes prepared from both thespleen and lymph nodes of the animal as fusion partners. The fused cellswere distributed into microculture plates and the hybridomas wereselected in DMEM supplemented with HAT and 15% horse serum. Hydridomasthat were found to produce antibodies to IFN-β2/IL-6-R were subcloned bythe limiting dilution method and injected into Balb/C mice that had beenprimed with pristane for the production of ascites. Immunoglobulins wereisolated from the ascites by ammonium sulfate precipitation (50%saturation), centrifuged, redissolved in water and dialyzed against PBScontaining 0.02% azide. The isotypes of the antibodies were definedeither with the use of a commercially available ELISA kit (Amersham,U.K.), or by the Ouchterlony method using commercially availableantisera against different isotypes.

The screening of the anti-IFN-β2/IL-6-R monoclonal antibodies producinghybridomas was performed as follow: Hybridoma supernatants were testedfor the presence of anti-IFN-β2/IL-6-R antibodies by an inverted solidphase radioimmunoassay (iRIA). PVC microtiter plates (DynatechLaboratories, Alexandria, VA) were coated with affinity purified goatanti-mouse serum F(ab)₂ antibodies (BioMakor) (10 μg/ml, 80 μl/well).Following overnight incubation at 4° C. the plates were washed twicewith PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked inwashing solution for at least 2 hrs at 37° C. Hybridoma culturesupernatants (50 μl/well) were added and the plates were incubated for 4hrs at 37° C. The plates were then washed three times with the washingsolution and ¹²⁵ I-IFN-β2/IL-6-R (50 μl, 10⁵ cpm) was added for furtherincubation of 16 hrs at 4° C. The plates were washed 3 times andindividual wells were cut and counted in a gamma counter. Samples givingcounts that were at least 5 times higher than the negative control valuewere considered positive (Table I). Thirty positive clones wereselected, subcloned for further studies and characterized.

EXAMPLE 5 Western blotting

Western blotting was performed as follows: Samples of partially purifiedIFN-β2/IL-6-R soluble fragment from human urine were analyzed by SDSPAGE under reducing conditions and electroblotted onto nitrocellulosesheets (BA85, Schleicher and Shuell). Following electroblotting thesheet was incubated overnight with a blocking buffer (5% non-fat milk inPBS containing 0.05% Tween 20 and 0.02% sodium azide) and then for 2 hrsat room temperature with the anti-IFN-β2/IL-6-R monoclonal antibody No.34-4. Following washing in 0.05% Tween 20 in PBS, ther nitrocellulosewas incubated for 4 hrs at room temperature with ¹²⁵ I-goat anti-mouseserum (0.7×10⁶ cpm/ml in the blocking buffer). The sheet was thenwashed, dried and autoradiographed.

Some of the isolated clones and subclones with their isotype and resultsof binding of IFN-β2/IL-6-R in inverted RIA and Western blotting arelisted in Table I.

                  TABLE I                                                         ______________________________________                                        Clones producing monoclonal antibodies to IFN-β2/IL-6 receptor                     Screening                                                                     with iRIA Western blot                                              Clone number                                                                              [CPM]       +M*     -M     Isotype                                ______________________________________                                        4.4         20,455      +       +      IgG.sub.1                              5            1,085      +       +      IgM                                    17.1        36,565      +              IgG.sub.2a                             20.2        31,450      +              IgG.sub.1                              22          11,465      +       +      IgG.sub.2                              24.2         8,850      +       +      IgG.sub.1                              25           2,000                     IgG.sub.2a                             28.7         1,645                     IgG.sub.1                              29           4,165                                                            30.8         1,755      +              IgM                                    31           3,060                                                            32.5        31,465      +       +      IgG.sub.1                              33.2        14,875                     IgG.sub.1                              34.4        33,480      +              k,IgG.sub.1                            35.2        35,495      +       +      k,IgG.sub.3                            36           1,445      +              IgM                                    37           9,640                     IgG.sub.1                              38.4        35,975      +              IgG.sub.1                              39.1         5,195      +       +      IgG.sub.2                              40           1,415      +              IgG.sub.1                              41           1,870      +              IgG.sub.1                              42.5        33,565                     IgG.sub.1                              43           1,255                     IgG.sub.1                              46           6,090                                                            48          18,000                     IgG.sub.1                              49           8,000      +              IgM                                    50.3        28,440      +       +      IgG.sub.1                              51           1,075                     IgG.sub.1                              52           3,945                     IgM                                    53.4         3,440                     IgG.sub.1                              ______________________________________                                         *M: mercaptoethanol (reducing agent)                                     

EXAMPLE 6 Affinity chromatography of IFN-β2/IL-6-R soluble fragmentpreparations with monoclonal antibodies

Antibodies against IFN-β2/IL-6-R are utilized for the purification ofthe soluble fragment by affinity chromatography. The monoclonal antibodyNo. 34-4 was used in this example for affinity chromatography, aftertesting its binding capacity for the radiolabeled antigen in inwertedsolid phase radioimmunoassay (iRIA). Ascitic fluid containing themonoclonal antibody secreted by hybridoma No. 34-4 was purified byammonium sulfate precipitation at 50% saturation followed by extensivedialysis against PBS. About 10 mg of immunoglobulins were bound to 1 mlpolyacrylhydrazide agarose as specified by Wilchek and Miron, Methods inEnzymology 34:72-76, 1979. 250 ml of human urine, partially purified oncarboxymethyl sepharose (CMS) column (equivalent to 250 l of crudeurine) were loaded on 0.5 ml of the anti-IFN-β2/IL-6-R antibody columnat 4° C. at a flow rate of 0.25 ml/min. The column was washed with PBSuntil no protein was detected in the washings. IFN-β2/IL-6-R solublefragment was eluted by 25 mM citric acid buffer, pH 2.2 (8×1 columnvolume fractions) and immediately neutralized by 1M Hepes buffer, pH8.5, with a total recovery of 88% IFN-β2/IL-6-R soluble fragment. Silverstain analysis of SDS PAGE of the eluted fractions revealed a major bandof M.W. of 50,000 and another major band of 150,000 (contaminants).Further purification of this preparation was obtained by RP-300 HPLC,and the soluble receptor fragment eluted at 39% acetonitrile in apattern similar to that of FIG. 1.

                                      TABLE II                                    __________________________________________________________________________    Immunoaffinity purification of IL-6 receptor from urine (CMS)                                         ELISA                                                 McAb       FLUORESCAMINE         purity                                                                            yield                                    column                                                                            Sample ml  ug/ml                                                                             ug   ug/ml                                                                             ug   %   %                                        __________________________________________________________________________    34.4                                                                              Load   250 2200                                                                              550,000                                                                            0.38                                                                              95                                                    Efluent                                                                              250 2000                                                                              500,000                                                                            0.06                                                                              15                                                    Elution 1                                                                            1.2  20 24   7.7 9    38                                               Elution 2                                                                            1.2  45 54   30.4                                                                              36.5 67                                               Elution 3                                                                            1.2  18   21.6                                                                             12  14.4 80                                               Elution 4                                                                            1.2  11 13   8   9.6  87                                               Total eluted            69.5     88                                       __________________________________________________________________________

EXAMPLE 7 ELISA test

Microtiter plates (Dynatech or Maxisorb by Nunc) were coated withanti-IFN-β2/IL-6-R monoclonal antibody No. 34-4 (immunoglobulinfraction, 120 μl, 20 μg/ml in PBS), overnight at 4° C. The plates werewashed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blockedin the same solution for at least 2 hrs at 37° C. The tested sampleswere diluted in the blocking solution and added to the wells (100μl/well) for 4 hrs at 37° C. The plates were then washed 3 times withPBS containing Tween 20 (0.05%) followed by the addition of rabbitanti-IFN-β2/IL-6-R serum (1:1000, 100 μl/well) for further incubation of4 hrs at 37° C. The plates were washed 3 times and a conjugate ofgoat-anti-rabbit horseradish peroxidase (HRP, BioMakor, 1:2000, 100μl/well) was added for 2 hrs at 37° C. The plates were washed 4 timesand the color was developed by ABTS (2,2'-azino-bus(3-ethylbenzthiazoline-6-sulfonic acid), Sigma) substrate. The plateswere read by an automatic ELISA reader. Alternatively, the ELISA couldbe performed by replacing the rabbit polyclonal anti-IFN-β2/IL-6-Rantibodies with a monoclonal antibody No. 22-1 either conjugated to HRPor biotinylated.

EXAMPLE 8

The activity of the IFN-β2/IL-6-R soluble extracellular fragment wastested on the hybridoma/plasmacytoma growth factor (HGF) activity ofIFN-β2/IL-6. Murine plasmacytoma T1165 cells were exposed for 16 hoursto a low concentration of pure human recombinant IFN-β2/IL-6, producedin E.coli and purified to homogeneity (2×10⁶ HGF units/mg). In parallel,the same IFN-β2/IL-6 samples were incubated for 90 minutes at 37° C.with various concentrations of the IFN-β2/IL-6-R soluble extracellularfragment and then added to the cells. With 0.25 ng/ml IFN-β2/IL-6 alone,there was no stimulation of ³ H-thymidine incorporation in the pulsedT1165 cells, but addition of IFN-β2/IL-6-R soluble extracellularfragment produced a dose-dependent stimulation (Table III). At 5 ng/mlIFN-β2/IL-6, growth stimulation of the T1165 cells was seen and onlyslightly stimulated by IFN-β2/IL-6-R soluble extracellular fragment. Asa control, the IFN-gamma soluble receptor purified from human urine hadno effect (Table III).

Subliminal concentrations of IFN-β2/IL-6 can, therefore, be specificallypotentiated by addition of IFN-β2/IL-6-R soluble extracellular fragment.Assuming about 5,000 IFN-β2/IL-6 receptors on the cells, 0.25 ng/mlIFN-β2/IL-6 corresponds to about 150 molecules of the cytokine perreceptor. The stimulatory effect of IFN-β2/IL-6-R soluble extracellularfragment begins to be seen when 5-10,000 IFN-β2/IL-6-R solubleextracellular fragment molecules are added per murine IFN-β2/IL-6receptor. Although part of the IFN-β2/IL-6-R soluble extracellularfragment may be inactive, it does not appear that the effect isstochiometric.

Several lines of human B-cells transformed by Epstein-Barr virus (EBV)show growth stimulation by IFN-β2/IL-6. In the TI cell line, addition ofIFN-β2/IL-6-R soluble extracellular fragment did not potentiate theeffect of IFN-β2/IL-6 as it did on the murine T1165 plasmacytoma (TableIV). In fact, with the TI cells, IFN-β2/IL-6-R soluble extracellularfragment behaved as an inhibitor of IFN-β2/IL-6 action at lowconcentrations of the cytokine.

In the HGF activity assay the bioactivity of the IFN-β2/IL-6 is measuredby stimulation of ³ H-thymidine incorporation in murine plasmacytomaT1165 cells (Nordan, R. P. and Potter, M. Science (1986) 233:566-568).Briefly, serial dilutions (60 μl) of the tested samples were incubatedwith T1165 cells (10⁴ cells in 40 μl) overnight at 37° C. in 96-wellmicrotiter plates. A pulse of ³ H-thymidine (1 μCi/well) was given for 4hrs at 37° C. Cells were harvested by an automatic harvester andcounted. One unit of IFN-β2/IL-6 is defined as the amount of proteinthat gives 50% of the maximal effect in the assay.

                  TABLE III                                                       ______________________________________                                        Stimulation of mouse plasmacytoma T1165 cell growth                                                       .sup.3 H-thymidine                                IFN-β2/IL-6                                                                       IFN-β2/IL-6-R soluble                                                                       incorporation                                     ng/ml    extracellular fragment                                                                           counts per minute                                 ______________________________________                                        None     None                5,800                                             0.25    None                5,780                                            "        0.03                7,750                                            "        0.06                9,100                                            "        0.12               10,100                                            "        0.25               20,000                                            "        0.50               29,750                                            "        1.00               40,400                                            2.5      None               16,000                                            2.5      0.25               16,000                                            5.0      None               32,000                                            5.0      0.25               48,000                                            R soluble fragment                                                                      6,350                                                               ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Stimulation of human EBV-transformed B-cell line TI                                      .sup.3 H-thymidine incorporation                                              counts per minute × 10.sup.-3                                IFN-β 2/IL-6    + IFN-β2/IL-6-R soluble                             ng/ml        Alone   extracellular fragment                                   ______________________________________                                        None         20      25                                                       0.05         33      25                                                       0.2          39      29                                                       12.5         39      40                                                       25.0         42      39                                                       ______________________________________                                         The IFNβ2/IL6-R soIuble extracellular fragment concentration was 0.6     μg/ml.                                                                

EXAMPLE 9 Activity on human breast cancer cells

Human breast ductal carcinoma cells T47D are growth-inhibited byIFN-β2/IL-6. These cells are rich in IFN-β2/IL-6 receptor binding sites,at least comparable to what is found on lymphocytes and myeloid cells. Aseries of subclones of the T47D cancer cell culture were isolated andcompared for their growth inhibition by IFN-β2/IL-6. By several tests,colony formation from sparsely seeded cells and ³ H-thymidineincorporation in semi-confluent monolayers, some clones (e.g. 07) weremore sensitive and others (e.g. 012) were less sensitive than theparental T47D cultures. The effect of IFN-β2/IL-6-R solubleextracellular fragment was tested in an ³ H-thymidine incorporationassay where IFN-β2/IL-6 produces a dose-dependent inhibition (Table V).

Whereas addition of IFN-β2/IL-6-R soluble extracellular fragment alonehad no effect, the soluble receptor strongly increased the antigrowtheffect of IFN-β2/IL-6 (Table V). Unlike what was found on the murineplasmacytoma cells, the effect of IFN-β2/IL-6-R soluble extracellularfragment on the human breast cancer cells was marked at higherIFN-β2/IL-6 concentrations. There was not only a reduction in the amountof IFN-β2/IL-6 needed for 50 percent inhibition, but the amplitude ofthe effect was markedly increased. In the presence of IFN-β2/IL-6-Rsoluble extracellular fragment, the inhibition of ³ H-thymidineincorporation reached 98 percent, while without IFN-β2/IL-6-R solubleextracellular fragment residual DNA synthesis was observed even at highIFN-β2/IL-6 doses. This result suggested that IFN-β2/IL-6-R solubleextracellular fragment may also make a more resistant subclone such as012 respond fully to IFN-β2/IL-6 growth inhibition. Indeed, addition ofIFN-β2/IL-6-R soluble extracellular fragment, allowed also to inhibitalmost completely the ³ H-thymidine incorporation in 012 cells (FIG. 3)and the effect was again evident at the higher IFN-β2/IL-6concentrations.

These results show that IFN-β2/IL-6-R soluble extracellular fragmentacts on human breast cancer cells and increases both the sensitivity andthe amplitude of the response.

                  TABLE V                                                         ______________________________________                                        Inhibition of Human Breast Carcinoma T47D clone 07 growth                                .sup.3 H-thymidine incorporation                                              counts per minute × 10.sup.-3                                IFN-β 2/IL-6    + IFN-β2/IL-6-R soluble                             ng/ml        Alone   extracellular fragment                                   ______________________________________                                        None         100     103                                                      br 1.5       57      32                                                       7.5          46      12                                                       36           37       4                                                       180          26       2                                                       900          12       4                                                       ______________________________________                                         The IFNβ2/IL6-R soluble extracellular fragment concentration was 0.6     μg/ml.                                                                

We claim:
 1. A process for the production of substantially purifiedIFN-β2/IL-6-R soluble extracellular fragment which comprises:(a)recovering the crude protein fraction from a dialyzed concentrate ofhuman urine; (b) subjecting said crude protein fraction of step (a) toaffinity chromatography on a column of immobilized IFN-β2/IL-6 oranti-IFN-β2/IL-6-R monoclonal antibody, to obtain affinity purifiedactive fractions of the IFN-β2/IL-6 binding protein; (c) applying saidaffinity purified active fractions from step (b) to reversed-phase highpressure liquid chromatography (HPLC); and (d) recovering the fractionscontaining substantially purified IFN-β2/IL-6-R soluble extracellularfragment, said protein having a molecular weight of about 50K on SDSPAGE under non-reducing conditions and moving as a single peak onreversed-phase HPLC.
 2. A process according to claim 1 wherein the crudeprotein fraction of step (a) is subjected to ion exchange chromatographyprior to the affinity chromatography of step (b).
 3. A process accordingto claim 1 wherein the affinity chromatography of step (b) is performedon a column of anti-IFN-β2/IL-6-R monoclonal antibody.
 4. A process inaccordance with claim 1, wherein said step (d) comprises recovering thefraction eluting at 39% acetonitrile.
 5. A process in accordance withclaim 1, wherein the affinity chromatography of step (b) is performed ona column of immobilized IFN-β2/IN-6.